Persister formation in Staphylococcus aureus is associated with ATP depletion

Conlon, Brian P.; Rowe, Sarah E.; Gandt, Autumn Brown; Nuxoll, Austin S.; Donegan, Niles P.; Zalis, Eliza A.; Clair, Gérémy; Adkins, Joshua N.; Cheung, Ambrose L.; Lewis, Kim

doi:10.1038/nmicrobiol.2016.51

Cited by 557 publications

(630 citation statements)

References 40 publications

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“…These results suggest that mutants with impaired metabolism and/or an inability to use different carbon sources might enter into a dormant state that is characteristic of the persister subpopulation (18,19). These results are supported by recent findings showing that the generation of persister cells is associated with depletion of ATP in Staphylococcus aureus (20).…”

supporting

confidence: 82%

Transposon-Sequencing Analysis Unveils Novel Genes Involved in the Generation of Persister Cells in Uropathogenic Escherichia coli

Molina-Quiroz

Lazinski

Camilli

et al. 2016

Antimicrob Agents Chemother

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c Persister cells are highly tolerant to different antibiotics and are associated with relapsing infections. In order to understand this phenomenon further, we exposed a transposon library to a lethal concentration of ampicillin, and mutants that survived were identified by transposon sequencing (Tn-Seq). We determined that mutations related to carbon metabolism, cell envelope (cell wall generation and membrane proteins), and stress response have a role in persister cell generation. Uropathogenic Escherichia coli (UPEC) is the main etiologic agent of urinary tract infections worldwide, generating approximately 80% of clinical cases every year. Most of these infections are chronic and represent a worldwide public health threat (1). Relapsing infections have been associated with the generation of persister cells (2, 3). This subpopulation is characterized by a transient nonhereditary dormant state that leads to survival in lethal concentrations of different antibiotics. The molecular mechanisms reported to be involved in the generation of persister cells include stochastic processes, toxin-antitoxin (TA) modules, and the stringent response (4-6). However, a deeper understanding of the physiological and genetic regulation of this process is lacking.Aiming to identify global regulators or metabolic pathways that E. coli might downregulate to enter into a dormant persister state, we used a high-throughput genetic screen to recognize mutants with increased fitness during exposure to a lethal concentration of the cell wall-active antibiotic ampicillin in order to identify novel regulators and/or metabolic pathways involved in the generation of persister cells. This method has previously been used to identify metabolic pathways associated with the generation of persister cells in stationary-phase cultures of E. coli exposed to gentamicin (7).We used a library of UPEC strain CFT073 containing approximately 360,000 random insertions of the EZ-Tn5 ϽR6K␥ori/ KAN-2Ͼ transposon (8). A 1:100 dilution of an overnight culture of the library was grown at 37°C in Luria-Bertani (LB) broth with aeration and when the optical density at 600 nm (OD 600 ) reached 0.3, the culture was split in two flasks, and ampicillin was added to a final concentration of 125 g ml Ϫ1 (10 times the MIC). After 6 h of incubation at 37°C with shaking, we enriched the survivors by overnight growth in fresh LB lacking ampicillin (approximately 45 generations of outgrowth).Total DNA, corresponding to three independent experiments, was extracted from the input and ampicillin-treated output cultures using a QIAamp DNA minikit (Qiagen).Transposon insertions in mutants showing increased survival in cultures exposed to ampicillin were identified by transposon sequencing (Tn-Seq) using the homopolymer tail-mediated ligation PCR (HTML-PCR) method followed by sequencing on a HiSeq 2500 system (Illumina) (9, 10). Briefly, DNA was fragmented by sonication, and poly(C) tails were added to all 3= termini using terminal deoxynucleotidyl transferase. Then, a nested PCR st...

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supporting

confidence: 82%

Transposon-Sequencing Analysis Unveils Novel Genes Involved in the Generation of Persister Cells in Uropathogenic Escherichia coli

Molina-Quiroz

Lazinski

Camilli

et al. 2016

Antimicrob Agents Chemother

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“…For example, a central role for the stringent response and toxin-antitoxin modules in Escherichia coli persistence has emerged (22,24), and similar mechanisms seem to be implicated in the persistence of P. aeruginosa (25) and Salmonella enterica serovar Typhimurium (26). Additionally, a mechanism for persister formation based on the depletion of ATP levels was recently described in S. aureus (27). However, it remains unclear to what extent these findings can be extrapolated to other Gramnegative or Gram-positive pathogens.…”

mentioning

confidence: 99%

In Vitro Emergence of High Persistence upon Periodic Aminoglycoside Challenge in the ESKAPE Pathogens

Michiels

Bergh

Verstraeten

et al. 2016

Antimicrob Agents Chemother

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bHealth care-associated infections present a major threat to modern medical care. Six worrisome nosocomial pathogens-Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.-are collectively referred to as the "ESKAPE bugs." They are notorious for extensive multidrug resistance, yet persistence, or the phenotypic tolerance displayed by a variant subpopulation, remains underappreciated in these pathogens. Importantly, persistence can prevent eradication of antibiotic-sensitive bacterial populations and is thought to act as a catalyst for the development of genetic resistance. Concentration-and time-dependent aminoglycoside killing experiments were used to investigate persistence in the ESKAPE pathogens. Additionally, a recently developed method for the experimental evolution of persistence was employed to investigate adaptation to high-dose, extended-interval aminoglycoside therapy in vitro. We show that ESKAPE pathogens exhibit biphasic killing kinetics, indicative of persister formation. In vitro cycling between aminoglycoside killing and persister cell regrowth, evocative of clinical high-dose extended-interval therapy, caused a 37-to 213-fold increase in persistence without the emergence of resistance. Increased persistence also manifested in biofilms and provided cross-tolerance to different clinically important antibiotics. Together, our results highlight a possible drawback of intermittent, high-dose antibiotic therapy and suggest that clinical diagnostics might benefit from taking into account persistence.

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“…These results implicate that vancomycin and ␤-lactams kill pneumococci by different mechanisms, although the LytA autolysin is required for the bactericidal activities of both vancomycin and penicillin (27,64). Alternatively, persisters in Gram-negative and -positive bacteria may be formed through different mechanisms, and/or behave differently in terms of tolerance efficacy to different bactericidal drugs, as evidenced in a recent study of persister cells in Staphylococcus aureus (22). Taken together, future investigations are warranted to decipher whether (i) induction of the ptv operon by a natural signal(s) confers vancomycin tolerance in wild-type pneumococci, (ii) the increased vancomycin tolerance observed in the ptv derepression/overexpression strains is related to LytA, (iii) induction of this operon is mechanistically related to the drug tolerance of typical persisters.…”

Section: Discussionmentioning

confidence: 71%

“…Many of these factors induce bacterial persistence by inhibiting translation through the activities of toxin-antitoxin (TA) systems (10,21). Gram-positive bacteria have also been documented to develop phenotypic tolerance to antibiotics (22), but the mechanisms are poorly understood. The GraSR two-component system is required for colistin-induced reversible tolerance of S. aureus to vancomycin (23).…”

mentioning

confidence: 99%

“…It is likely that GraSR can regulate the surface charge of S. aureus via D-alanylation of teichoic acids and lysinylation of phosphatidylglycerol and thus enable bacterial resistance to cationic antimicrobial peptides (24,25). Also, ATP depletion is associated with persister formation in S. aureus (22). LexA, a DNA-binding protein that represses the SOS response in bacteria, is involved in the formation of Streptococcus mutans persisters induced by the competence-stimulating peptide (CSP) (26).…”

mentioning

confidence: 99%

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Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae

Feng

et al. 2017

J Bacteriol

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Reversible or phenotypic tolerance to antibiotics within microbial populations has been implicated in treatment failure of chronic infections and development of persister cells. However, the molecular mechanisms regulating phenotypic drug tolerance are largely unknown. In this study, we identified a four-gene operon in Streptococcus pneumoniae that contributes to phenotypic tolerance to vancomycin (ptv). RNA sequencing, quantiative reverse transcriptase PCR, and transcriptional luciferase reporter experiments revealed that transcription of the ptv operon (consisting of ptvR, ptvA, ptvB, and ptvC) is induced by exposure to vancomycin. Further investigation showed that transcription of the ptv operon is repressed by PtvR, a PadR family repressor. Transcriptional induction of the ptv operon by vancomycin was achieved by transcriptional derepression of this locus, which was mediated by PtvR. Importantly, fully derepressing ptvABC by deleting ptvR or overexpressing the ptv operon with an exogenous promoter significantly enhanced vancomycin tolerance. Gene deletion analysis revealed that PtvA, PtvB, and PtvC are all required for the PtvR-regulated phenotypic tolerance to vancomycin. Finally, the results of an electrophoretic mobility shift assay with recombinant PtvR showed that PtvR represses the transcription of the ptv operon by binding to two palindromic sequences within the ptv promoter. Together, the ptv locus represents an inducible system in S. pneumoniae in response to stressful conditions, including those caused by antibiotics.IMPORTANCE Reversible or phenotypic tolerance to antibiotics within microbial populations is associated with treatment failure of bacterial diseases, but the underlying mechanisms regulating phenotypic drug tolerance remain obscure. This study reports our finding of a multigene locus that contributes to inducible tolerance to vancomycin in Streptococcus pneumoniae, an important opportunistic human pathogen. The vancomycin tolerance phenotype depends on the PtvR transcriptional repressor and three predicted membrane-associated proteins encoded by the ptv locus. This represents the first example of a gene locus in S. pneumoniae that is responsible for antibiotic tolerance and has important implications for further understanding bacterial responses and phenotypic tolerance to antibiotic treatment in this and other pathogens.KEYWORDS Streptococcus pneumoniae, phenotypic tolerance, vancomycin, PadR family regulator, gene regulation, transcriptional derepression

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Persister formation in Staphylococcus aureus is associated with ATP depletion

Cited by 557 publications

References 40 publications

Transposon-Sequencing Analysis Unveils Novel Genes Involved in the Generation of Persister Cells in Uropathogenic Escherichia coli

Transposon-Sequencing Analysis Unveils Novel Genes Involved in the Generation of Persister Cells in Uropathogenic Escherichia coli

In Vitro Emergence of High Persistence upon Periodic Aminoglycoside Challenge in the ESKAPE Pathogens

Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae

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